Bhat et al (Diabetologia 2013, 56, 1417-1424), Bhat et al. (Biochem Pharmacol. 2013, 85, 1655-62), Gault et al. (J Biol Chem. 2013, 288, 35581-91) as well as Finan et al. (Nat Med. 2015, 21, 27-36) described trigonal agonists of the glucagon-like peptide-1 (GLP-1), the glucagon and the glucose-dependent insulinotropic polypeptide (GIP) receptors, e.g. by combining the actions of GLP-1, glucagon and GIP in one molecule, which leads to a therapeutic principle with anti-diabetic action and a pronounced weight lowering effect superior to pure GLP-1 agonists, among others due to glucagon-receptor mediated increased satiety and energy expenditure as well as GIP receptor mediated increased insulin secretion.
The amino acid sequence of GLP-1(7-36)-amide is shown as SEQ ID NO: 1.
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2
Liraglutide is a marketed chemically modified GLP-1 analog in which, among other modifications, a fatty acid is linked to a lysine in position 20 leading to a prolonged duration of action (Drucker et al, Nat. Rev. Drug Disc. 2010, 9, 267-268; Buse et al., Lancet 2009, 374, 39-47).
The amino acid sequence of Liraglutide is shown as SEQ ID NO: 2.
HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4-hexadecanoylamino-butyryl)EFIAWLVRGRG-OH
Glucagon is a 29-amino acid peptide which is released into the bloodstream when circulating glucose is low. Glucagon's amino acid sequence is shown as SEQ ID NO: 3.
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH
During hypoglycemia, when blood glucose levels drop below normal, glucagon signals the liver to break down glycogen and release glucose, causing an increase of blood glucose levels to reach a normal level. Recent publications suggest that glucagon has in addition beneficial effects on reduction of body fat mass, reduction of food intake, and increase of energy expenditure (Heppner et al., Physiology & Behavior 2010, 100, 545-548).
GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acid peptide that is released from intestinal K-cells following food intake. GIP and GLP-1 are the two gut enteroendocrine cell-derived hormones accounting for the incretin effect, which accounts for over 70% of the insulin response to an oral glucose challenge (Baggio et al. Gastroenterology 2007, 132, 2131-2157).
GIP's amino acid sequence is shown as SEQ ID NO: 5.
YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH
Peptides which are based on the structures of GLP-1 or glucagon, and bind and activate the GLP-1, the glucagon and the GIP receptor have been described in patent applications WO 2010/011439, WO 2010/148089, WO 2012/088116, WO 2013/192129, WO 2013/192130, WO 2014/049610 and WO 2015/067716. Further trispecific agonists based on exendin-4 have been described in WO 2014/096145, WO 2015/086731, WO 2015/086732, WO 2015/086733, WO2015/155141, and PCT/EP2016/063332. The compounds described therein have been shown to lead to improved glycemic control, possible islet and beta-cell preservation and enhanced body weight loss.
Peptides which bind and activate both the GIP and the GLP-1 receptor designed as analogues of exendin-4 and substituted with a fatty acid side chain are described in patent applications WO 2014/096145 A1, WO 2014/096150 A1, WO 2014/096149 A1, and WO 2014/096148 A1.
Exendin-4 is a 39 amino acid peptide which is produced by the salivary glands of the Gila monster (Heloderma suspectum. Exendin-4 is an activator of the GLP-1 receptor, whereas it shows low activation of the GIP receptor and does not activate the glucagon receptor (see Table 1).
TABLE 1Potencies of exendin-4 at human GLP-1, GIP and Glucagonreceptors (indicated in pM) at increasing concentrationsand measuring the formed cAMP as described in Methods.SEQ IDEC50 hGLP-EC50 hGIPEC50 hGlucagonNO:Peptide1R [pM]R [pM]R [pM]4exendin-40.412500.0>10000000
The amino acid sequence of exendin-4 is shown as SEQ ID NO: 4.
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2
Exendin-4 shares many of the glucoregulatory actions observed with GLP-1. Clinical and nonclinical studies have shown that exendin-4 has several beneficial antidiabetic properties including a glucose dependent enhancement in insulin synthesis and secretion, glucose dependent suppression of glucagon secretion, slowing down gastric emptying, reduction of food intake and body weight, and an increase in beta-cell mass and markers of beta cell function.
These effects may be beneficial not only for diabetics but also for patients suffering from obesity. Patients with obesity have a higher risk of getting diabetes, hypertension, hyperlipidemia, cardiovascular and musculoskeletal diseases.
Relative to GLP-1, exendin-4 is resistant to cleavage by dipeptidyl peptidase-4 (DPP4) resulting in a longer half-life and duration of action in vivo (Eng J., Diabetes, 1996, 45 (Suppl 2):152A (abstract 554)).
Exendin-4 was also shown to be much more stable towards degradation by neutral endopeptidase (NEP), when compared to GLP-1, glucagon or oxyntomodulin (Druce et al., Endocrinology, 2009, 150(4), 1712-1722). Nevertheless, exendin-4 is chemically labile due to methionine oxidation in position 14 (Hargrove et al., Regul. Pept., 2007, 141, 113-119) as well as deamidation and isomerization of asparagine in position 28 (WO 2004/035623).
Bloom et al. (WO 2006/134340) disclose that peptides which bind and activate both the glucagon and the GLP-1 receptor can be constructed as hybrid molecules from glucagon and exendin-4, where the N-terminal part (e.g. residues 1-14 or 1-24) originates from glucagon and the C-terminal part (e.g. residues 15-39 or 25-39) originates from exendin-4. Such peptides comprise glucagon's amino acid motif YSKY in position 10-13. Krstenansky et al (Biochemistry, 1986, 25, 3833-3839) show the importance of these residues 10-13 of glucagon for its receptor interactions and activation of adenylate cyclase.
Compared to GLP-1, glucagon and oxyntomodulin, exendin-4 has beneficial physicochemical properties, such as solubility and stability in solution and under physiological conditions (including enzymatic stability towards degradation by enzymes, such as DPP4 or NEP), which results in a longer duration of action in vivo.
Nevertheless, also exendin-4 has been shown to be chemically labile due to methionine oxidation in position 14 as well as deamidation and isomerization of asparagine in position 28. Stability might be further improved by substitution of methionine at position 14 and the avoidance of sequences that are known to be prone to degradation via aspartimide formation, especially Asp-Gly or Asn-Gly at positions 28 and 29.